WO2024015531A2 - Lipides triazines, synthèse de lipides et procédés d'inhibition de l'activité transcriptionnelle de nf kb canonique - Google Patents

Lipides triazines, synthèse de lipides et procédés d'inhibition de l'activité transcriptionnelle de nf kb canonique Download PDF

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WO2024015531A2
WO2024015531A2 PCT/US2023/027667 US2023027667W WO2024015531A2 WO 2024015531 A2 WO2024015531 A2 WO 2024015531A2 US 2023027667 W US2023027667 W US 2023027667W WO 2024015531 A2 WO2024015531 A2 WO 2024015531A2
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lipid
triazine
lipids
viral
canonical
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WO2024015531A3 (fr
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Vincent J. Venditto
David NARDO PADRON
Abdullah Al MASUD
Julian Agustin MORY
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University Of Kentucky Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D251/00—Heterocyclic compounds containing 1,3,5-triazine rings
    • C07D251/02—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings
    • C07D251/12—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D251/26—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hetero atoms directly attached to ring carbon atoms
    • C07D251/40—Nitrogen atoms
    • C07D251/54—Three nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids

Definitions

  • the presently disclosed subject matter relates to lipids which can be administered with an immunostimulatory antigen. More particularly, the presently disclosed subject matter relates to triazine lipids which can be utilized in non-viral vectors for administration with immunostimulatory antigens, and which inhibit canonical NFKB transcriptional activity during an immune response. Methods for synthesizing triazine lipids are also describe herein.
  • BACKGROUND [0003] Liposomes provide an optimal vehicle for pharmaceutical delivery due to their versatility as amphipathic vectors, which allows for delivery of hydrophobic and hydrophilic agents. 1,2 By altering the lipid composition in these nanoparticles, a multitude of properties can be honed to optimize their functionality.
  • liposome research has fueled the development of synthetic lipids to improve therapeutic delivery, particularly nucleic acids. 3
  • the complexity and cost of novel lipids limits liposome research. 1,4–6
  • various research groups have developed synthetic, cationic lipid libraries with the goal of improving siRNA and mRNA delivery using cost effective and high-throughput schemes, taking advantage of specific chemical structures that allow for rapid headgroup diversification. 5–7 [0004]
  • liposomes have been investigated extensively for vaccine development using nucleic acids or proteins 8,9 both as adjuvants, 10 and as anchors of antigens on liposomal surfaces.
  • lipid-based vaccines have been investigated extensively as immunomodulators since haptenated lipids were first formulated in 1974.
  • 129, 130 The modular format of lipid delivery systems provides a platform for inclusion of hydrophobic or hydrophilic adjuvants in a nanoparticle to increase antigen retention at sites of injection, improve immune recognition, and immune cell uptake.
  • SARS-CoV-2 vaccines as well as approved subunit vaccines for influenza, malaria, shingles, and human papilloma virus (HPV), with several additional formulations in clinical development.
  • the adjuvants currently approved by the United States Federal Drug Administration (FDA) for use alone or in lipid-based vaccines include alum, monophosphoryl lipid A (MPLA), cytosine phosphoguanine (CpG), saponins, squalene, and combinations of each.
  • 131-133 Adjuvant induced immune response can be characterized both by the robustness of immune responses and the TH1/TH2 balance as measured by antibody profiles (e.g. IgG2a/IgG1).
  • 134, 135 TH1 responses are primarily classified as cell-mediated immunity, and opsonizing antibodies (e.g. IgG2a) are a marker of this response.
  • TH2 responses are typically classified as humoral responses, and antibodies induced for protection against extracellular pathogens (e.g. IgG1) mark this response.
  • Overactive TH1 responses can cause tissue damage and uncontrolled TH2 responses can cause allergic responses.
  • a balanced, antigen-specific TH1/TH2 response represents the ideal vaccine induced immune profile.
  • Saponin e.g. QS-21
  • Alum is skewed heavily toward a TH2 response, while MPLA, CpG, and squalene are skewed heavily toward a TH1 response.
  • the adjuvants included in lipid-based vaccines target specific innate immune pathways associated with robust immune responses.
  • the underlying premise builds from the concept that nanoparticles containing adjuvants are recognized and taken up by antigen presenting cells, stimulated through pattern recognition receptors (PRRs) that leads to activation of B cells and T cells.
  • PRRs pattern recognition receptors
  • the resultant immune response associated with these formulations is marked by both protective immunity and a reactogenic response associated with “flu-like” symptoms.
  • Such undesirable responses result in subjects who prefer not to be vaccinated.
  • Vaccine-induced reactogenicity is most commonly observed side-effect of vaccination with increased levels of pyrogenic cytokines (e.g. IL-6, TNF ⁇ ) that promote pain, swelling and redness at the sight of injection as well as headache, fever, myalgia, and fatigue. 140 Therefore, strategies that promote protective immunity using small molecule immune modulators, while limiting reactogenicity, are desirable.
  • the presently disclosed subject matter includes a triazine lipid.
  • the triazine lipid is of the formula , wherein: R1 is .
  • R1 comprises at least 12 alkyl carbons.
  • R 1 comprises 18 alkyl carbons.
  • R 1 comprises 12 alkyl carbons.
  • R2 and R3 each comprise .
  • the triazine lipid is .
  • the triazine lipid i .
  • the presently disclosed subject matter also includes a non-viral triazine lipid-based vector including a plurality of triazine lipids consistent with one or more of the above-identified lipids.
  • the presently disclosed subject matter also includes a solid-phase synthesis method for synthesizing triazine lipids. In the method, a resin is reacted with a first amine headgroup to generate an amine terminated resin. In some embodiments, the resin is an amine-reactive resin. A dichlorotriazine is then formulated substituting the amine terminated resin with a cyanuric chloride via nucleophilic aromatic substitution.
  • the presently disclosed subject matter also includes a method for inhibiting canonical NFKB transcriptional activity during an immune response to an immunostimulatory antigen within a subject.
  • the method includes administering a non-viral triazine lipid-based vector including a plurality of triazine lipids to a subject concurrently with an immunostimulatory antigen.
  • the immunostimulatory antigen is an immunogenic peptide. In some embodiments, the immunostimulatory antigen is an immunostimulatory nucleic acid. In some embodiments, each lipid of the plurality of triazine lipids is cationic. In some embodiments, the triazine lipid includes a lipid tail group, a triazine linker, and a cationic headgroup. In some embodiments, the lipid tail comprises a saturated or unsaturated dialkylamine. In some embodiments, each triazine lipid of the non-viral vector is of the formula , wherein: R1 is alkyl; . In some embodiments, R 1 comprises 18 or fewer alkyl carbons.
  • R 1 comprises at least 12 alkyl carbons. In some embodiments, wherein R 1 comprises 18 alkyl carbons. In some embodiments, R1 comprises 12 alkyl carbons.
  • the non-viral triazine lipid-based vector is a liposome. In some embodiments the non-viral triazine lipid-based vector administered to the subject may include one or more additional lipids selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), distearolyphosphatidycholine (DSPC), and 1, 2-distearoyl-sn-glycero-3- phsphoethanolamine-polyethylene glycol (DSPE-PEG). In some embodiments, the non-viral triazine lipid-based vector administered in the method may stimulate non-canonical NF K B transcriptional activity. In one such embodiment, each triazine lipid of the plurality of triazine
  • the immunostimulatory antigen is an immunogenic polypeptide and the non-viral triazine-based vector invokes an anti-polypeptide response which is greater than an anti-polypeptide response invoked by dioleoyl-3-trimethylammonium propone (DOTAP) or 1,2-Dimyristoyl-sn-glycero-3- phosphocholine (DMPC) when administered concurrently with the immunogenic polypeptide.
  • DOTAP dioleoyl-3-trimethylammonium propone
  • DMPC 1,2-Dimyristoyl-sn-glycero-3- phosphocholine
  • the non-viral triazine lipid-based vector and the immunostimulatory antigen may be administered together as an immunogenic composition including the non-viral triazine lipid-based vector and the immunostimulatory antigen.
  • the presently disclosed subject matter also includes a method for inhibiting canonical transcriptional activity during an immune response within one or more cells.
  • the method includes contacting the one or more cells with one or more triazine lipids.
  • FIG.1 shows synthetic schemes for producing triazine (TZ) lipids.
  • FIG.2A is a graph showing the transition temperature of TZ lipids determined by DSC.
  • FIG.2B is a graph showing in vitro toxicity of triazine lipids. Toxicity of TZ lipids on bone marrow-derived macrophages as compared to commercially available cationic (DOTMA) and zwitterionic (DMPC) lipids using the lactate dehydrogenase assay. Liposomes were made by thinfilm hydration followed by sonication and used immediately to treat cells for 24 hours, prior to testing LDH release in cell media.
  • DOTMA commercially available cationic
  • DMPC zwitterionic
  • FIG.3 shows the efficacy of TZ lipids in gene transfection.
  • A Gel shift assay of plasmid DNA complexed with TZ lipids.
  • B pKa assessment of cationic lipids measured by TNS fluorescence at pH range 2.5 to 10. Plots represent the sigmoidal, bestfit analysis of one of three independent experiments.
  • C–E Transfection of HeLa cells with luciferase reporter gene using Lipofectamine 3000 or TZ lipids at an N : P ratio of 10, 5 and 2.5 (left to right).
  • Bars represent the mean values from one of three representative experiments, except for the LDH assay which was performed twice.
  • C Luciferase expression in transfected HeLa cells.
  • D LDH release from HeLa cells transfected with luciferase plasmid 4 hours after transfection.
  • E Viability of cells treated with plasmid and lipids 24 hours after transfection.
  • F–H Transfection of HEK293-T cells with hAAT using Lipofectamine 3000 or TZ lipids at N : P ratios of 6, 3 and 1.5 (left to right). Bars represent the mean values from one of three representative experiments, except for the viability assay which was performed twice.
  • Liposomal vaccines can include various components, including natural phospholipids and adjuvants, to optimize responses to an immunogen.
  • FIG.5 shows that TZ3 does not induce significant in vivo toxicity at 20 mM. Seven- week-old C57BL/6J mice were administered 100 ⁇ L of 20 mM cationic lipid (TZ3, TZ9 or DOTAP (Do)) nanoparticles intraperitoneally in HEPES buffered saline.
  • (A) Serum alanine aminotransferase (ALT), (B) Interleukin 6 (IL-6), and (C) serum creatinine (SCr) levels were measured 48 hours after treatment. Fold-change from baseline measurements drawn one week prior were compared with those of untreated animals (NT). Lines represent mean, and dots represent individual animals. Equal numbers of each sex were included; however, the TZ9 group represents only the 7 surviving animals. Significance was compared using one way ANOVA and Dunnett’s (A) or Kruskal Wallis (B and C) tests; only significant comparisons are shown.
  • FIG.6 shows that PEG550, DOPE, and TZ3 improve transfection efficiency with LNPs, but LPs exhibit improved transfection efficiency and reduced toxicity in vivo.
  • A-B HEK293T cells were transfected with 200 ng GFP plasmid per well using LNPs and analyzed three days later for GFP expression by flow cytometry.
  • A LNPs formulated with 50% TZ3, 10% DSPC, 39% cholesterol and 1% DSPE-PEG(550-2000), or 40% cholesterol and no PEG.
  • B LNPs formulated with 50% DOTAP (Do) or TZ3, 10% DSPE or DOPE, 39% cholesterol and 1% DSPE-PEG550.
  • C-F Male and female BALB/c mice were administered 1 x 109 genome copies of AAV8-GFP or 10 ⁇ g of GFP plasmid in either LNPs made with 50% TZ3, 10% DOPE, 39% cholesterol and 1% DSPE-PEG550 or LPs made with 50% TZ3 and 50% DOPE.
  • hepatocytes were evaluated for: (C) percent GFP positive cells, or (D) mean fluorescence intensity (MFI).
  • E Percent weight change and (F) serum ALT were also evaluated at the same time point.
  • FIG.7 shows that TZ3 LP transfection is more efficient in vivo than TZ3 LNPs or formulations made with DOTAP.
  • mice were administered 10 ⁇ g of hAAT DNA with LNPs made with 50% TZ3 or DOTAP (Do), 10% DOPE, 39% cholesterol and 1% DSPE- PEG550 or LPs made with 50% TZ3 and 50% DOPE. Seventy-two hours later, protein expression in the serum was assessed via ELISA. Lines represent mean hAAT concentration; dots represent individual animals. Data was compared with Kruskal-Wallis test. [0023] FIG.8 shows that transgene expression using TZ3 as a delivery vector elicits minimal antibody responses, while administration of hAAT protein with TZ3 results in significant immunogenicity.
  • mice were administered 10 ⁇ g of hAAT DNA with LNPs made with 50% TZ3 or DOTAP (Do), 10% DOPE, 39% cholesterol and 1% DSPE-PEG550 or LPs made with 50% TZ3 and 50% DOPE; or 25 ⁇ g of hAAT protein in saline or 1 mM lipid solution.
  • RET anti-hAAT IgG reciprocal endpoint titers
  • FIG.9 shows that PEGylation decreases LNP uptake by antigen presenting cells.
  • A Bone marrow-derived dendritic cells (DC) or J774 macrophages were incubated for 18 hours with LNPs made with 5% DiD and DSPE-PEG2000, or PEG-free liposomes.
  • FIG.10 shows intermediate compounds A-G for the synthesis of triazine lipids.
  • FIG.11 shows 1 H Nuclear Magnetic Resonance (NMR) for intermediate compound.
  • FIG.12 shows 13 C NMR for intermediate compound A.
  • FIG.13 shows 1 H NMR for intermediate compound B.
  • FIG.14 shows 13 C NMR for intermediate compound B.
  • FIG.15 shows 1 H NMR for 2-[(Triphenylmethyl)thio]ethanamine.
  • FIG.16 shows 13 C NMR for 2-[(Triphenylmethyl)thio]ethanamine.
  • FIG.17 shows 1 H NMR for intermediate compound C.
  • FIG.18 shows 13 C NMR for intermediate compound C.
  • FIG.19 shows 1 H NMR for intermediate compound D.
  • FIG.20 shows 13 C NMR for intermediate compound D.
  • FIG.21 shows the 1 H NMR for intermediate compound E.
  • FIG.22 shows 13 C NMR for intermediate compound E.
  • FIG.23 shows 1 H NMR for intermediate compound F.
  • FIG.24 shows the 13 C NMR for intermediate compound F.
  • FIG.25 shows 1 H NMR for intermediate compound G.
  • FIG.26 shows 13 C NMR for intermediate compound G.
  • FIG.27 shows 1 H NMR for lipid 1 (TZ1).
  • FIG.28 shows 13 C NMR for TZ1.
  • FIG.29 shows 1 H NMR for lipid 2 (TZ2).
  • FIG.30 shows 13 C NMR for TZ2.
  • FIG.31 shows 1 H NMR for lipid 3 (TZ3).
  • FIG.32 shows 13 C NMR for TZ3.
  • FIG.33 shows 1 H NMR for lipid 4 (TZ4).
  • FIG.34 shows 13 C NMR for TZ4.
  • FIG.35 shows 1 H NMR for lipid 5 (TZ5).
  • FIG.36 shows 13 C NMR for TZ5.
  • FIG.37 shows 1 H NMR for lipid 6 (TZ6).
  • FIG.38 shows 13 C NMR for TZ6.
  • FIG.39 shows 1 H NMR for lipid 7 (TZ7).
  • FIG.40 shows 13 C NMR for TZ7.
  • FIG.41 shows 1 H NMR for lipid 8 (TZ8).
  • FIG.42 shows 13 C NMR for TZ8.
  • FIG.43 shows 1 H NMR for lipid 9 (TZ9).
  • FIG.44 shows the 13 C NMR for TZ9.
  • FIG.45 shows 1 H NMR for lipid 10 (TZ10).
  • FIG.46 shows 13 C NMR for TZ10.
  • FIG.47 shows 1 H NMR for lipid 11 (TZ11).
  • FIG.48 shows 13 C NMR for TZ11.
  • FIG.49 shows 1 H NMR for lipid 12 (TZ12).
  • FIG.50 shows 13 C NMR for TZ12.
  • FIG.51 shows HPLC traces of lipids TZ1-TZ4 and chloroform (used as solvent), detected at 205 and 254 (254 shown). The mobile phase was a gradient of water and acetonitrile with 0.1% trifluoroacetic acid, as indicated, and constant 5% methanol with 01% trifluoroacetic acid. *Shortened due to speed of compound elution.
  • FIG.52 shows HPLC traces of lipids TZ5-8 and chloroform (used as solvent), detected at 205 and 254 (254 shown).
  • the mobile phase was a gradient of water and acetonitrile with 0.1% trifluoroacetic acid, as indicated, and constant 5% methanol with 01% trifluoroacetic acid.
  • the four compounds corresponding to TZ5-8 needed a mixture of isopropanol and chloroform for proper dissolution of HPLC.
  • FIG.53 shows HPLC traces of lipids TZ9-TZ12 and chloroform (used as solvent), detected at 205 and 254 (254 shown).
  • FIG.54 shows HPLC traces of free apolipoprotein A-I and apolipoprotein A-I lipopeptide.
  • FIG.55 shows in vivo toxicity of TZ3 and TZ9 at 10 mM. Seven-week-old C57BL/6J mice were administered 100 ⁇ L of 10 mM cationic lipid (TZ3, T9, or DOTAP(Do)) intraperitoneally.
  • FIG.56 shows extent of mice transfection with hAAT plasmid administered in lipid nanoparticles including TZ3 or DOTAP (Do).
  • FIG.57 shows (A) Four weeks after LP transfection, protein expression in the serum was assessed via ELISA. Only values above the limit of quantification are shown.
  • FIG.58 shows schemes for flow cytometry analysis.
  • A Scheme for GFP quantification in HEK293T cells stained with Zombie NearIR Dye after transfection with GFP plasmid with LNPs.
  • FIG.59 is a diagram showing the canonical and non-canonical pathways for NF K B stimulation. NFKB stimulation is mediated through two distinct pathways (canonical and non- canonical), which are both regulated by CIAP1/2 in the basal state.
  • FIG.60 shows cationic triazine-based lipid (TZ3) induces an increase greater than 3 orders of magnitude in antibody titter toward a model antigen in the absence of additional adjuvants with a balanced TH1/TH2 response. Titers are shown for sham immunized mice, protein administered in saline, protein administered with DOTAP (DO), or protein administered with cationic triazine lipid (TZ3).
  • FIG.61 is a schematic showing thermally controlled aromatic substitution of cyanuric chloride.
  • FIG.62 shows (A) structures of triazine lipids used in toxicity experiments, (B) quantified by lactate dehydrogenase activity as a marker of early apoptosis. Bone marrow derived macrophages were incubated with lipids for 24 hours at decreasing concentrations (250, 125, 62.5, 31.5 ⁇ M). Media and triton are used as negative and positive controls, respectively.
  • DO dioctadecenyl-3-trimethylammonium propane
  • DMPC dimyristoylphosphatidylcholine.
  • FIG.63 shows the inhibition of canonical NFKB activity in human THP-1-Blue cells stimulated with TNF ⁇ (shown) or LPS (not shown) using cationic triazine-based lipids containing primary amine headgroups (TZ3, TZ4).
  • IC50 values TZ3, 4 ⁇ M; DOTAP, 28 ⁇ M; TZ4, 29 ⁇ M.
  • NFKB inhibition is quantified after 24h lipid treatment.
  • FIG.64 is a diagram showing a solid-phase synthesis method for synthesizing a triazine lipid library.
  • FIG.65 shows J774 murine macrophages treated with cationic triazine lipid (TZ3) for 16 hours exhibit does-dependent increase in mean fluorescence intensity (MFI) of CD80 relative to untreated cells.
  • FIG.66 shows TZ3 inhibition of canonical NF K B activity is dose dependent, but not affected by duration of exposure. As shown, NFKB inhibition is comparable at 1-9h.
  • the term “immunostimulatory antigen” refers to a protein, peptide, or other molecule or macromolecule, in whole or in part, capable of eliciting an immune response.
  • the term “immunostimulatory antigen” can refer to, in some embodiments, an immunogenic polypeptide, and, in other embodiments, an immunostimulatory nucleic acid.
  • the term “immunogenic polypeptide” refers to a polypeptide which, when introduced to a target cell, invokes a protective immune response, such as an inflammatory response or induction of cytokines.
  • a protective immune response such as an inflammatory response or induction of cytokines.
  • polypeptide can refer to, in some embodiments, a polypeptide and, in other embodiments, a protein.
  • an “immunogenic polypeptide” can be, in some embodiments, a polypeptide which invokes an immune response , and, in other embodiments, a protein which invokes an immune response.
  • the term “immunostimulatory nucleic acid” refers to a molecule of nucleotides which encodes for an immunogenic polypeptide or which otherwise invokes or enhances an immune response.
  • the immunostimulatory nucleic acid may be a deoxyribonucleic acid (DNA) molecule, while, in other embodiments, the immunostimulatory nucleic acid may be a ribonucleic acid (RNA) molecule (e.g., mRNA or siRNA).
  • an effective amount of in the context of inhibiting canonical Nuclear Factor Kappa B (NF K B) transcriptional activity during an immune response with a non-viral triazine lipid-based vector refers to an amount of the non-viral triazine lipid-based vector, which, when administered to the subject, inhibits a reactogenic response within the subject induced by an immunostimulatory antigen, such as an immunogenic polypeptide or an immunostimulatory nucleic acid.
  • an effective amount of the non- viral triazine lipid-based vector may be administered concurrently with the immunostimulatory antigen, e.g., as part of a vaccine or other immunogenic composition.
  • the term “subject” can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
  • the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • the subject is a mammal.
  • a “patient” refers to a subject afflicted with a disease or disorder.
  • patient includes human and veterinary subjects.
  • the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, and would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds.
  • the term “pharmaceutically-acceptable carrier” refers to a solid or liquid filler, diluent, and/or encapsulating substance that may be safely administered to a subject to facilitate delivery of a composition.
  • the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration, percentage, or the like is meant to encompass variations of in some embodiments ⁇ 50%, in some embodiments ⁇ 40%, in some embodiments ⁇ 30%, in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
  • ranges can be expressed as from “about” one particular value, and/or to “about” another particular value.
  • the presently disclosed subject matter includes triazine lipids which may be utilized as non-viral transfection vectors for administration with an immunostimulatory antigen.
  • a triazine lipid is of the formula , wherein: .
  • R 1 comprises 18 or fewer alkyl carbons.
  • R 1 comprises at least 12 alkyl carbons.
  • R 1 comprises 18 alkyl carbons.
  • R1 comprises 12 alkyl carbons.
  • R2 and R 3 each comprise .
  • the triazine lipid is
  • the triazine lipid is or .
  • the presently disclosed subject matter also includes non-viral triazine lipid-based vectors including a plurality of triazine lipids, where each lipid is of a formula consistent with that specified above. Accordingly, in some embodiments, each lipid of the plurality of triazine lipids of a vector may be of the formula , wherein: R1 is alkyl; R2 . In some embodiments, R1 of each triazine lipid of the vector comprises 18 or fewer alkyl carbons. In some embodiments, R 1 of each triazine lipid of the vector comprises at least 12 alkyl carbons.
  • R1 of each triazine lipid of the vector comprises 18 alkyl carbons. In some embodiments, R1 of each triazine lipid of the vector comprises 12 alkyl carbons. In some embodiments, R 2 and R 3 of each triazine lipid of the vector each comprise . In some embodiments, each triazine lipid of the vector is
  • the each triazine lipid of the vector is [00109]
  • the presently disclosed subject matter also includes solid-phase synthesis method for synthesizing triazine lipids, some or all of which, may be used as non-viral vectors for administration with an immunostimulatory antigen.
  • a resin such as an amine- reactive resin
  • a first amine headgroup to generate an amine terminated resin.
  • the amine-reactive resin is 2-chlorotrityl chloride resin.
  • a dichlorotriazine is then formulated substituting the amine terminated resin with a cyanuric chloride via nucleophilic aromatic substitution.
  • the dichlorotriazine is then reacted with a lipid tail to form a monochlorotriazine, which is subsequently reacted with a second amine group to form the triazine lipid.
  • the triazine lipid is cleaved from the resin.
  • the amine-reactive resin is 2-chlorotrityl chloride resin.
  • the lipid tail is a saturated or unsaturated dialkylamine. In one such embodiment, the lipid tail is selected from the group consisting of , and .
  • the first amine headgroup is a diamine.
  • the first amine headgroup is selected from the group consisting of In some embodiments, the second amine headgroup is selected from the group consisting of In some embodiments, the second amine headgroup is a diamine. It is appreciated, that the triazine lipids which can be synthesized employing the above-described method of synthesis is not necessarily limited limited to those including amine headgroups and tail groups expressly referred to above.
  • alternative headgroups with at least one reactive nucleophilic amine which can be substituted with cyanuric chloride via nucleophilic aromatic substitution to yield a dichlorotriazine or added to a tailed monochlorotriazine in a manner consistent with that disclosed above may, in some embodiments, be alternatively utilized.
  • alternative lipid tails with at least one reactive nucleophilic amine which can be added to the dichlorotriazine in a manner consistent with that disclosed above may, in some embodiments, be alternatively utilized.
  • the lipid tail may contain primary or secondary amines and may be unsaturated and/or branched.
  • the presently disclosed subject matter also includes methods for inhibiting canonical NF K B transcriptional activity during an immune response to an immunostimulatory antigen within a subject.
  • the method includes administering a non-viral triazine lipid-based vector including a plurality of triazine lipids to a subject concurrently with an immunostimulatory antigen.
  • the immunostimulatory antigen is an immunogenic peptide.
  • the immunostimulatory antigen is an immunostimulatory nucleic acid.
  • each lipid of the triazine lipids is cationic.
  • the triazine lipid includes a lipid tail group, a triazine linker, and a cationic headgroup.
  • the lipid tail comprises a dialkylamine.
  • each triazine lipid of the non-viral vector is of the formula , wherein: R1 is alkyl; R2 is
  • the non-viral triazine lipid-based vector is a liposome. Liposomes including triazine lipids disclosed herein may be formed by any suitable method. For example, in some embodiments, the liposomes may be formed by thin film hydration. In some embodiments, thin film hydration may be followed by sonication. In some embodiments, the non-viral triazine lipid-based vector is lipid nanoparticles.
  • Lipid nanoparticle vectors including triazine lipids disclosed herein may be formed by any suitable method.
  • lipid nanoparticle vectors may be formed via thin film hydration.
  • the non-viral triazine lipid-based vector administered to the subject in the method may include one or more additional lipids selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), distearolyphosphatidycholine (DSPC), and 1, 2-distearoyl-sn-glycero-3-phsphoethanolamine-polyethylene glycol (DSPE-PEG).
  • DOPE dioleoylphosphatidylethanolamine
  • DSPC distearolyphosphatidycholine
  • DSPE-PEG 1, 2-distearoyl-sn-glycero-3-phsphoethanolamine-polyethylene glycol
  • the non-viral triazine lipid-based vector includes DSPE-PEG(550-2000). In some embodiments, the non-triazine lipid-based vector administered in the method may stimulate non-canonical NF K B transcriptional activity. In one such embodiment, each triazine
  • the immunostimulatory antigen is an immunogenic polypeptide and the non-viral triazine-based vector invokes an anti-polypeptide response which is greater than an anti- polypeptide response invoked by dioleoyl-3-trimethylammonium propone (DOTAP) or 1,2- Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) when administered concurrently with the immunogenic polypeptide.
  • DOTAP dioleoyl-3-trimethylammonium propone
  • DMPC 1,2- Dimyristoyl-sn-glycero-3-phosphocholine
  • the anti-polypeptide response with the non- viral triazine lipid-based vector is greater than the anti-polypeptide response invoked by DOTAP or DMPC.
  • the anti-polypeptide response with the non-viral triazine lipid- based vector is at least 300-fold greater relative to the anti-polypeptide response with DOTAP.
  • the non-viral triazine lipid-based vector and the immunostimulatory antigen may be administered together as an immunogenic composition including the non-viral triazine lipid-based vector and the immunostimulatory antigen.
  • the non-viral triazine lipid-based vector and the immunostimulatory antigen are administered with a pharmaceutically-acceptable carrier.
  • Certain triazine lipids described herein may also find utility in modulating NF K B pathways in vitro.
  • the presently disclosed subject matter also includes a method for inhibiting canonical transcriptional activity during an immune response within one or more cells.
  • the one or more cells are contacted with one or more triazine lipids.
  • each triazine lipid of the one or more triazine lipids is cationic.
  • Each triazine lipid of the one or more triazine lipids may be of the formulation and include the feature specified for the various embodiments of the triazine lipids of the triazine lipid-based vector described above with respect to the methods for inhibiting canonical NFKB transcriptional activity during an immune response to an immunostimulatory antigen within a subject.
  • Vangasseri, et al. 175 utilized CD80 expression as one marker of activity in cells treated with DOTAP.
  • CD80 expression was enhanced with increasing concentrations of DOTAP, which Vangasseri, et al. attribute to TLR9 signaling and canonical NF ⁇ B stimulation, but may actually be the result of non-canonical NF ⁇ B activation.
  • Vangasseri, et al. demonstrate that anionic lipids (e.g. PA, PG) and zwitterionic lipids (e.g.
  • PC zwitterionic lipids that are capped with an ethyl group on the phosphate rendering them cationic (e.g. EPC) result in increased CD80 expression.
  • EPC ethyl group on the phosphate rendering them cationic
  • Vangasseri, et al. demonstrate that lipids with transition temperatures below 50 °C (e.g. DM, DO) exhibit increased CD80 expression relative to their high Tm counterparts (e.g. DS, DP). Therefore, based on this observation there is an indication of promiscuity in the cationic lipid head group for inclusion in the lipid, while lipid tails shorter than C16, and those with unsaturation, or branching that result in reduced transition temperature will have improved activity.
  • headgroups of the triazine lipids utilized in the vectors and methods disclosed herein may sometimes be referred to as including a specific amine headgroup or selected from a particular grouping of candidate amine headgroups and/or including a specific saturated, unbranced tail or selected from a particular grouping of saturated tails, unbranched tails
  • triazine lipids including alternaterative cationic lipid headgroups and/or unsaturated or branching tails which cause the triazine lipid to exhibit a transition temperature below 50 °C can, in some embodments, be alternatively used in the vectors and method disclosed herein.
  • Example 1 In vitro Reagents Synthesized Using Cyanuric Chloride [00115] A library of triazine (TZ) based lipids was synthesized using cyanuric chloride as a linker, dialkylamines as lipid tails and various small molecules to generate diverse head groups. The headgroups were chosen due to their cost effectiveness, commercial availability, and diversity in functional moieties, which provide a platform for future evaluation of an expanded library of triazine based lipids.
  • TZ triazine
  • Beta-alanine-tert-butyl ester, cyanuric chloride, didodecyl- amine, diisopropylethylamine (DIPEA), 2-mercaptoethylamine HCl, morpholine, ninhydrin, N,N- dimethyl diaminopropane and trityl chloride were purchased from TCI America (Portland, OR).
  • Dioctadecylamine was purchased from Sigma-Aldrich (Milwaukee, WI).
  • N-Boc-1,3- diaminopropane was purchased from Matrix Scientific (Columbia, SC).1,2-Dimyristoyl-sn- glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- dimyristoyl-sn-glycero-3-phospho-(10-rac-glycerol) (DMPG), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and cholesterol were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL).
  • DMPC 1,2-Dimyristoyl-sn- glycero-3-phosphocholine
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DOPE 1,2- dimyristo
  • Solvents for reactions were purchased from various suppliers through VWR (Radnor, PA). Thin layer chromatography (TLC; Millipore Sigma, Silica gel 60 F254) was visualized under UV light or with 2% ninhydrin in DMSO. Final compound purity was assessed via a Waters 2707 Autosampler, Waters 2545 Quaternary Gradient Module pump and Waters 2998 Photodiode Array Detector following injection into a Waters XBridge C183.5 mm column (part no. 186003034) using a water, acetonitrile and methanol mixture as described in the figures below and detected at 205 and 254 nm.
  • mice Six-week-old female C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in a specific pathogen-free facility at the University of Kentucky. Mice were sedated for immunization and blood collection with isoflurane gas. Blood was collected by superficial temporal vein puncture using a small animal lancet (Medipoint) into a microcentrifuge tube and centrifuged for 10 min at 2000 x g after standing at 4°C for 1h. Serum was stored at -80 °C for later antibody detection. All procedures were approved by the University of Kentucky Institutional Animal Care and Use Committee.
  • the lipid tail was reacted first to form a dichloro-triazine, followed by headgroup diversification through addition of various nucleophilic small molecule moieties as headgroups.
  • the first NAS was initiated on ice and allowed to stir at room temperature in chloroform for at least 4 hours.
  • the second substituent was added at room temperature in chloroform and heated to 50 °C for at least 24 hours.
  • the final NAS reaction was performed in xylenes or dioxane and heated from room temperature to 80 °C for at least 72 hours. In each reaction, excess nucleophile or DIPEA served as base.
  • the reactions were monitored at each step via thin layer chromatography and characterized by nuclear magnetic resonance and mass spectrometry.
  • Intermediate A was prepared by adding 1 equiv. of cyanuric chloride to a stirring solution of chloroform with 2.4 equiv. of beta-alanine-tert-butyl ester and 10 equiv. of DIPEA on ice. The mixture was allowed to come to room temperature, then heated overnight at 50 °C. Remaining beta-alanine-tert-butyl ester was removed by washing the dried product three times with brine.
  • Intermediate B was prepared by adding 1 equiv. of cyanuric chloride to a stirring solution of chloroform with 2.4 equiv. of N-Boc-1,3-diaminopropane and 10 equiv. of DIPEA on ice. The mixture was allowed to come to room temperature, then heated overnight at 50 °C. Remaining N-Boc-1,3-diaminopropane was removed by washing the dried product three times with brine.
  • Lipid 1 was prepared by adding 1 equiv. of didodecylamine to a stirring solution of dioxane containing 2 equiv. of intermediate A and 10 equiv. of DIPEA. The solution was heated to 80 °C. After at least 48 hours (shorter reaction periods led to reduction in product yield) the reaction was evaporated using a rotary evaporator and re-dissolved in chloroform then washed three times with brine. The organic phase was then dried over magnesium sulfate, filtered, and dried in a rotary evaporator.
  • Lipid 3 was prepared by adding 1 equiv. of didodecylamine to a stirring solution of dioxane containing 2 equiv. of intermediate B and 10 equiv. of DIPEA. The solution was heated to 80 °C. After at least 48 hours (shorter reaction periods led to reduction in product yield) the reaction was evaporated and dissolved in chloroform then washed three times with brine. The organic phase was then dried over magnesium sulfate, filtered, and dried using a rotary evaporator.
  • Lipid 5 was prepared by adding 1 equiv. of didodecylamine to a stirring solution of dioxane containing 2 equiv. of intermediate C and 10 equiv. DIPEA. The solution was heated to 80 °C. After at least 48 hours (shorter reaction periods led to reduction in product yield) the reaction was concentrated by rotary evaporation and re-dissolved in chloroform then washed three times with brine.
  • the organic phase was then dried over magnesium sulfate, filtered, and dried using a rotary evaporator.
  • the resulting solid was deprotected using a mixture of 1:1 TFA in dichloromethane and evaporated to dryness.
  • the resulting solid was purified by silica gel chromatography by first eluting impurities with chloroform and ethyl acetate, then eluting the final product with methanol. The methanol fraction was dried and re-dissolved in chloroform before being filtered over magnesium sulfate to yield lipid 5 (90.6%, final product).
  • Lipid 8 was prepared by adding 1 equiv. of intermediate E to 8 equiv. of morpholine in xylenes or dioxane and heating to 80 °C for 48 hours. The solvent was removed using a rotary evaporator at 80-90°C and the resulting solid was dissolved in chloroform and washed three times with 0.5 M HCl then twice with brine. The organic phase contained a number of impurities and was purified by silica gel chromatography using at 0-10% ethyl acetate:chloroform mobile phase gradient.
  • Lipid 9 was prepared by adding 20 equiv. of N,N-dimethyl-1,3- diaminopropane to a stirring solution of intermediate F and 10 equiv. of DIPEA in dioxane. The reaction was allowed to stir at room temperature for 24 hours then heated at 80 °C for another 48 hours. The reaction was then concentrated using a rotary evaporator and the product was dissolved in ethyl acetate and washed three times with brine.
  • Lipid 11 was prepared by adding 4-8 equiv. of N-Boc-1,3- diaminopropane to a stirring solution of dioxane containing 1 equiv. of intermediate G and 10 equiv. of DIPEA. The solution was stirred at 80 °C for 72 hours after which the solvent was removed using a rotary evaporator. The resulting solid was then dissolved in chloroform and washed three times with 0.5 M HCl then twice with brine.
  • Lipid nanoparticles for all experiments were formed by thin lipid film hydration.
  • the triazine lipids dissolved in chloroform were transferred in sufficient quantities (based on the desired final concentration and volume) into a sterile round bottom tube alone or in combination with other lipids (DOPE, DSPC, cholesterol, etc.).
  • DOPE, DSPC, cholesterol, etc. The organic solvents were to form a thin lipid film and dried overnight under vacuum.
  • the lipids were then rehydrated in 20 mM HEPES solution and sonicated at 60 °C until opalescent ( ⁇ 30 min).
  • Tm transition temperature of the lipids was determined using a multicell differential scanning calorimeter (TA Instruments).
  • Liposomes were made with triazine lipids at a concentration of 10 mM in 20 mM HEPES buffer. These were heated to 60 °C and sonicated until the solution was translucent. For T m determination, 250 ⁇ L of the liposome solution was transferred into reusable hastelloy ampoules while 250 ⁇ L of the HEPES solution was transferred to the third ampoule, leaving the reference ampoule empty. For lipids 7 and 8, which failed to form nanoparticles, 250 ⁇ L of the solution containing the lipid aggregate were transferred to the ampoules after sonication. Data was collected over a range of 10-110 °C at a rate of 2 °C min -1 in a heat-cool-heat cycle.
  • the CpCalc 2.1 software package was used to convert the raw data into a molar heat capacity and the data from the second heating cycle were processed using Microsoft Excel.
  • Size and Charge Determination The size and charge of the nanoparticles were determined using a Zetasizer Nano ZS (Malvern Panalytical). For each lipid, two separate formulations of liposomes were tested at a concentration of 1 mM. The size was determined in ZEN0400 cuvettes using the following settings: four measurements of 15 five second runs detected at a backscatter angle of 173° at 25 °C.
  • the zeta potential for the liposomes was determined in a DTS1070 folded capillary zeta cell using the following settings; four measurements of at least 50 runs modelled with the Smoluchowski equation at 25 °C using the automatic settings from the instrument.
  • Carboxyfluorescein Encapsulation Assay The ability of CC lipids to encapsulate molecules was tested using 5-(6)-carboxy-fluorescein (CF) purchased from Acros Organics (Pittsburg, PA), which was purified using the protocol established by Ralston et al. 17 Briefly, unpurified CF was dissolved in refluxing ethanol for 3 hours in the presence of activated charcoal and filtered.
  • CF 5-(6)-carboxy-fluorescein
  • the filtrate was diluted in enough distilled water to achieve a 1 : 2 ethanol/water ratio and crystalized at 20 °C.
  • the crystalized CF was filtered and washed multiple times with distilled water and dried overnight. Solid CF was then dissolved in water and 5 M NaOH to a concentration of 250 mM and passed over an LH-20 Sephadex column. Five mL were purified on a 10 x 2 cm column by elution at room temperature with distilled water. CF eluted as a dark orange-red band that was quantified via absorbance at 492 nm using a coefficient of 6-CF (76900 M cm -1 ) as described by Weinstein et al.
  • liposomes from the various cationic lipids were rehydrated in a solution of 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate and 130 mM NaCl at a pH range of 2.5 to 12.
  • the pH of each formulation was reassessed to ensure that the pH had not significantly deviated from the original solution and 180 ⁇ L of each formulation was mixed with 20 ⁇ L of 10 ⁇ M TNS in distilled water (for a final TNS concentration of 1 ⁇ M).
  • the solutions were mixed by pipetting and incubated at room temperature for 10 minutes, before being analysed for fluorescence intensity using a 321 nm excitation and 445 nm emission wavelengths.
  • BMDM mature bone marrow-derived macrophages
  • lipids concentration denoted in figure legend
  • HEPES buffer as negative control
  • Triton X-100 as positive control.
  • the 96 well plates were centrifuged at 200 x g for 5 minutes to remove debris and 100 ⁇ L of media was transferred to an untreated flat-bottom 96 well plate.
  • 100 ⁇ L of LDH reaction reagent purchased from Cayman Chemical (Ann Arbor, MI) was added to each and allowed to sit for 30 minutes at 37 °C.
  • Nanoparticles consisting of a 1 : 1 molar ratio of cationic lipid/DOPE were rehydrated in a 20 mM HEPES solution at pH 4. The nanoparticles were mixed at equal volumes (5 ⁇ L) with plasmid DNA (5 ⁇ L) at the amine to phosphate (N : P) ratios indicated in the figure legends and incubated at room temperature for 10 minutes.
  • the amine quantity per lipid was assumed to be 2 (one per headgroup), while DOTMA was considered to have 1 amine per lipid.
  • 10 ⁇ L of the lipoplex was mixed with 2 ⁇ L of 6 loading dye (Boston BioProducts) and loaded onto a 1% agarose gel containing 0.5 ⁇ g mL -1 of ethidium bromide and run at 100 mV for 60 minutes. The gels were visualized and photographed using a Bio-Rad ChemiDoc XR system using the manufacturer's software.
  • HeLa cells cultured in EMEM (ATCC) supplemented with 10% fetal bovine serum were transferred to a 96 well plate, in quadruplicate, at a density of 20,000 cells per well and incubated for 24 hours at 37 °C in 5% CO2.
  • Liposomes made with a 1 : 1 ratio of DOPE and TZ lipid were added to 200 ng pGL3 Luciferase Reporter Vector (Promega) at N : P ratios of 2.5, 5 and 10, and incubated at 37 °C for 10 minutes before being diluted in 100 ⁇ L of nonsupplemented EMEM and added to the cells.
  • a cell culture lysis reagent at pH 7.8 composed of 25 mM Tris–phosphate buffer, 0.7 g L- 1 1,2-diaminocyclohexane, 10% glycerol, 1% Triton X-100, and 1% protease inhibitor cocktail (Millipore).
  • a cell culture lysis reagent at pH 7.8 composed of 25 mM Tris–phosphate buffer, 0.7 g L- 1 1,2-diaminocyclohexane, 10% glycerol, 1% Triton X-100, and 1% protease inhibitor cocktail (Millipore).
  • Total protein content was determined with a bicinchoninic acid assay (G-Biosciences) and luciferase protein expression was quantified by a luciferase assay (Promega).
  • HEK293-T cells were seeded, in triplicate, on 24 well plates at a density of 50000 cells per well using D-MEM containing 10% fetal bovine serum (Gemini), 100 U mL -1 penicillin, 0.1 mg mL -1 streptomycin and 500 ⁇ g mL -1 geneticin (VWR) and incubated until they reached 70–90% confluency.
  • D-MEM fetal bovine serum
  • VWR 500 ⁇ g mL -1 geneticin
  • Lipoplexes were formed by combining TZ lipid liposomes made with a 1 : 1 ratio of DOPE and TZ lipid in Opti-MEM (Thermo) with 400 ng of human alpha-1 antitrypsin (hAAT) plasmid DNA (Addgene no.126704) and incubating for 12 minutes in Opti-MEM, before being added to cells. After 24 hours the media was removed for evaluation of viability and replaced with fresh media. The cells were then incubated for another 72 hours and then transferred to 1.5 mL microcentrifuge tubes and centrifuged at 400 rpm for 5 minutes.
  • hAAT human alpha-1 antitrypsin
  • the plate was then washed with 200 ⁇ L of phosphate buffered saline with 0.1% Tween-20 (PBS–T) four times and blocked with 100 ⁇ L of PBS with 0.05% casein (Beantown Chemical, 124240; PBS–C) for 1 hour at 37 °C.
  • the plate was then washed again, and 100 mL of fresh media from cells were plated, in duplicate, along with a standard curve made by serially diluting purified hAAT (OriGene no. RG202082) in PBS–C from 50 ng mL -1 to 0.048 ng mL and incubated for 1 hour at 37 °C.
  • mouse anti-hAAT monoclonal IgG2a antibody R&D Systems no. MAB1268-SP
  • HRP conjugated goat anti-mouse IgG2a (Abcam no.98698) was added at a 1 : 5000 dilution and incubated for 30 minutes at 37 °C.
  • Liposomal immunizations were administered subcutaneously to three groups (n 1 ⁇ 4 5 per group) of eight-week-old female C57BL/6J mice (The Jackson Laboratory) housed in a specific pathogen-free facility at the University of Kentucky.
  • the immunization, administered at 8 and 10 weeks of age, consisted of 50 ⁇ L of a 20 mM liposomal formulation prepared with a mixture of DMPC, DMPG, cholesterol, and monophosphoryl lipid A (MPL; Sigma) at a 15 : 2 : 3 : 0.3 molar ratio and 0.5 mg mL -1 of lipid- conjugated peptide.
  • the peptide used for these experiments was the lecithin–cholesterol acyltransferase domain of apolipoprotein A-I (sequence ⁇ AGGLSPVAEEFRDRMRTHVDSLRTQLAPHSEQMRESLAQRLAELKSN).
  • the original peptide anchor (cholesterylhemisuccinate) was used to immunize one group of mice, while two other groups were immunized with the peptide was conjugated to intermediate D and the third group was immunized with peptide free liposomes.
  • mice were sedated during any procedures using isoflurane gas. All animal procedures were performed in accordance with the United States Department of Health and Human Services, Office of Laboratory Animal Welfare, Public Health Service Policy on Humane Care and Use of Laboratory Animals and approved by the University of Kentucky Institutional Animal Care and Use Committee. [00158] Apolipoprotein A-I Peptide Titer ELISA.
  • Biotinylated apolipoprotein A-I peptide was diluted to a concentration 2 ⁇ g mL -1 in phosphate buffered saline with 0.1% Tween-20 (PBS–T) and plated in a 96-well streptavidin-coated plate (Thermo Fisher No.05124) using a volume of 100 ⁇ L. The peptide was incubated for 2 h at 37 °C, then washed six times with 200 ⁇ L of PBS–T.
  • Mouse serum (100 ⁇ L) was serially diluted in phosphate buffered saline containing 0.05% casein (PBS–C; Beantown Chemical) in duplicate, starting at 1 : 200 and incubated for 30 minutes at 37 °C. The wells were then washed six times and treated with 100 ⁇ L of goat anti-mouse IgG–HRP (Invitrogen no.16066) diluted 1 : 2000 in PBS–C and incubated for 30 minutes at 37 °C before being washed again. Binding was quantified by incubating the samples with 100 mL of tetramethylbenzidine (Rockland) for 30 minutes at room temperature, followed by quenching with 0.5 M H2SO4.
  • PBS–C phosphate buffered saline containing 0.05% casein
  • cyanuric chloride undergoes nucleophilic aromatic substitution at 0 °C for the first substitution, 25 °C for the second, and 70 °C for the third but the reactions are influenced by the nucleophilicity and steric hinderance of the reactants.
  • two dichlorotriazine molecules were generated as the basis for lipids and several small molecules were tested as headgroups (FIGS.1 and 10).
  • Lipids 1–4 proceeded well under both routes with similar yields for the convergent and divergent route using the beta-alanine headgroups (lipid 2: 20% and 21%) and the diaminopropane headgroups (lipid 4: 48% and 53%). This was not the case for lipids 5 and 6, which employed trityl-protected cysteamine (Trt-Cys). Divergent synthesis of lipidated dichlorotriazine molecules with Trt-Cys resulted in an insoluble compound with exceedingly low yield and could only be successfully synthesized using the convergent route with protected beta- alanine as the first substitution on cyanuric chloride.
  • the divergent route facilitated synthesis and purification of the final lipids.
  • the divergent synthetic route reduces the total number of reactions needed to prepare a library of molecules by 25–33% depending on the final composition of the lipids. While some lipids result in similar overall yield between convergent and divergent routes, the challenges with nucleophilicity, steric hinderance and purification of intermediate molecules resulted in synthetic preference for one route over the other for certain headgroups.
  • the divergent route results in increased compositional diversity with fewer steps, while the convergent route serves as an important complementary role for the synthesis of certain lipids.
  • Lipids 7 and 8 which contained morpholine in the headgroup, failed to form liposomes, alone or in combination with distearoyl phosphatidylcholine (DSPC) or DSPC and cholesterol from 5 to 90 mol% TZ lipids.
  • Other lipids made with isonipecotic acid also failed to form liposomes (data not shown) indicating that steric hinderance of the headgroups may preclude liposome formation.
  • lipids 11 and 12 initially formed nanoparticles, the structures were unstable past 24 hours as determined by dynamic light scattering. Aside from these, all other lipids formed nanoparticles that appear stable at one month after preparation when stored in the refrigerator, based on dynamic light scattering.
  • lipids that formed nanoparticles ranged from 87 to 186 nm in diameter (Table 1), with no clear trend between diameter and structural characteristics, such as lipid tail and charge.
  • Lipids containing cysteamine in the headgroup achieved the smallest size, while lipids 9 and 10, exhibited the largest initial diameter.
  • Lipids 11 and 12 also formed nanoparticles >300 nm in diameter, but are unstable as denoted in Table 1 with PDI of 0.98 and 0.51, respectively.
  • the charges of each formulation also aligned with the headgroup used and ranged from -75 to 70 mV for anionic and cationic headgroups, respectively.
  • Lipids 11 and 12 which contained beta-alanine and 1,3-diaminopropane in the headgroup, were hydrated in acidic conditions yielding a positive charge for this formulation. Attempts were made to formulate these in both acidic (pH 4) and basic (pH 10) solutions, but the lipids only hydrated well in acidic conditions. [00171] Prior to testing the nanoparticles in further applications, the toxicity of the compounds was assessed in vitro. The primary mechanisms of toxicity associated with lipid nanoparticles, particularly cationic ones, are cell lysis and activation of immune responses.
  • macrophages were chosen to test this aspect of TZ nanoparticles as these are among the primary cells responsible for the uptake of nanoparticles from circulation and are associated with the immune responses observed following in vivo nanoparticle administration.
  • the lipids were tested for induction of lactate dehydrogenase (LDH) release from bone marrow derived macrophages (BMDMs) from C57BL/6J mice, which has been demonstrated to be a more sensitive method of early liposomal toxicity.
  • LDH lactate dehydrogenase
  • BMDMs bone marrow derived macrophages
  • 25 BMDMs were treated with TZ lipids at concentrations ranging from 31.25 to 250 nmol mL -1 .
  • the toxicity of the nanoparticles ranged between that of the synthetic, cationic lipid DOTMA, and the natural zwitterionic phospholipid DMPC (Table 1).
  • lipid nanoparticles made from a 1 : 1 molar ratio of cationic lipids and DOPE were incubated with plasmid DNA at increasing ratios of cationic amine (N) to anionic nucleic acid phosphate (P) and assessed for migration in an agarose gel.
  • Formulations with a 1 : 1 molar ratio of DOPE and cationic lipid have been extensively reported in the literature and provide a simple starting point for assessing the potential of cationic lipid formulations. 29,30
  • the N content of TZ lipids are based on the distal aliphatic amines of the headgroups, but the other amines in the molecules may contribute to complexation.
  • FIG.3A all four lipids were able to complex RNA at an N : P ratio of 5 or above.
  • DOTMA/DOPE nanoparticles inhibited RNA migration at an N : P ratio of 10 while pure DOPE lipids were unable to prevent migration.
  • the TZ/DOPE lipoplexes were also evaluated by DLS at N : P ratios of 1 and 5 and their size and charge was determined by DLS. As evidenced in Table 2, the lipoplexes increased in size, compared with the free nanoparticles, but they increased slightly in size as more DNA was added, suggesting complexation.
  • pKa is another crucial aspect of cationic lipids that has been directly correlated to the success of lipid nanoparticles in gene delivery. 19,31,32 Particularly, ionizable lipids with a pKa ranging from 6.2 to 6.4, have been shown to achieve a high degree of efficacy when used to deliver siRNA.
  • HEK293-T cells were transfected with a plasmid encoding human alpha-1 antitrypsin (hAAT) using the same lipid mixtures, and hAAT expression was assessed by ELISA.
  • hAAT human alpha-1 antitrypsin
  • FIGS.3F–3H the cationic TZ lipids significantly improved pDNA transfection, except for lipid 10, and exhibited a similar toxicity profile to that of Lipofectamine.
  • the data from these experiments suggests that TZ lipids may have utility in the delivery of nucleic acids and warrants further exploration of their capabilities in vivo.
  • mice were immunized twice with a liposomal vaccine containing one of the lipopeptide conjugates (FIG.4B) or a control formulation without peptide present and reciprocal endpoint titers (RET) toward the peptide immunogen were assessed seven days after the second immunization.
  • a liposomal vaccine containing one of the lipopeptide conjugates (FIG.4B) or a control formulation without peptide present and reciprocal endpoint titers (RET) toward the peptide immunogen were assessed seven days after the second immunization.
  • RET reciprocal endpoint titers
  • Example 1 Because the in vitro evaluation described above in Example 1 was based on the use of lipoplexes (LPs) it was decided to also compare these to lipid nanoparticles (LNPs), as these are reported to have improved efficacy in vivo. 59 Formulations were developed using the lipids displayed in Table 3 based on standard DOTAP formulations described previously. Table 3. Structure of triazine lipids and other lipids used in plasmid formulations. [00182] However, further optimization of the formulations with triazine lipids was required, leading to several novel findings.
  • LPs lipoplexes
  • LNPs lipid nanoparticles
  • the present disclosure describes the ability of optimized TZ lipid formulations to improve in vivo plasmid transfection beyond that of standard DOTAP formulations as well as the immunologic response targeting the transgenes using each formulation.
  • the present disclosure also reveals that lipid nanoparticles and lipoplexes including certain TZ lipid formulations induce minimal antibody profiles toward a transgene delivered therewith while serving as a platform to induce robust antibody responses when used to directly deliver a protein. Accordingly, the present disclosure thus evidences the use of TZ-based lipids as non-viral vectors for gene delivery.
  • LNPs are denoted as circles, while LPs are square and protein injections are triangles.
  • mice and Cells [00185] Mice were purchased from Jackson Labs at 5-6 weeks of age and used in experiments at 7-9 weeks. C57BL/6J (#000664) mice were used for toxicity experiments shown in FIGS.5 and 55, which is incorporated herein by reference, while BALB/cJ (#000651) were used for transfections in all other figures. Equal numbers of male and female mice were used in each experiment. Mice were sedated using isoflurane gas prior to blood collection by saphenous vein puncture or intraperitoneal (i.p.) injections. Baseline serum levels of all experimental parameters were established one week prior to injections.
  • HEK293T cells kindly donated by Dr. Gregory Graf of the University of Kentucky College of Pharmacy, J774A.1 macrophages (ATCC TIB-67) or bone marrow derived dendritic cells were used for cell experiments and maintained at 37 °C with 5% CO2.
  • BMDCs Bone Marrow Derived Dendritic Cells
  • Lipid Nanoparticles Two types of nanoparticles were used for experiments: liposomes and plasmid lipid nanoparticles (LNPs). In both cases, the lipids used were dissolved in chloroform, mixed at the ratio described in each figure legend, dried into a thin lipid film by rotary evaporation, and placed under house vacuum overnight before use.
  • liposomes To form liposomes, the dried lipids were rehydrated in HEPES buffered saline (20 mM HEPES, 145 mM NaCl, pH 7) (HBS) with a pH of 4 and sonicated until translucent at 60 ⁇ C before being mixed with HBS to the final concentration and pH 7.4.
  • HBS HEPES buffered saline
  • Lipoplexes were formed from liposomes by mixing liposomes and DNA at a ratio of 6:1 positive to negative charges in Opti-MEM (for cells) or HBS (for mice) and incubated at room temperature for at least 12 minutes prior to administration.
  • LNPs dried lipids were rehydrated to a concentration of 10 mM in ethanol with 10 ⁇ L of 5 M HCl per mL of ethanol and mixed with a solution of DNA at 40 ng/ ⁇ L of DNA in 300 mM citric acid, pH 4.
  • the ethanol and aqueous solutions were mixed into LNPs using the Ignite microfluidic system (Precision NanoSystems) at a ratio of 1:3 ethanol to aqueous, at a rate of 12 mL/min.
  • Nanoparticle size was determined using a Zetasizer Nano ZS (Malvern Panalytical) with the following settings: four measurements of fifteen, five second runs detected at a backscatter angle of 173° at room temperature.
  • the zeta potential for the liposomes was determined in a DTS1070 folded capillary zeta cell using the following settings: four measurements of at least 50 runs modelled with the Smoluchowski equation at room temperature using the automatic settings from the instrument.
  • the concentration of DNA in LNPs after dialysis was quantified using an AccuClear® Ultra High Sensitivity dsDNA Quantification Kit (Biotium #31027) and quantified on a BioTek Synergy H1 plate reader. Encapsulation efficiencies were determined by comparing the amount of DNA in the LNP solution vs.
  • mice were administered 0.1 mL of the liposomal solution i.p.48 hours later, the mice were bled for evaluation of serum creatinine (SCr; Crystal Chem no.80350), alanine aminotransferase (ALT; AAT Bioquest no.13803) and interleukin-6 (IL-6; Biolegend no. 431304) according to manufacturer instructions.
  • SCr Crystal Chem no.80350
  • ALT alanine aminotransferase
  • IL-6 interleukin-6
  • mice were administered 200 ⁇ L nanoparticles or PBS vehicle i.p. at the doses indicated in the figure legend. Seventy-two hours after injection the mice were bled again for assessment of ALT levels and hAAT expression. hAAT levels were determined via ELISA using serum diluted 1:1 in PBS containing 0.05% casein (PBS-C; 124250; Beantown Chemical), as described previously.
  • PBS-C casein
  • mice were administered GFP plasmid (Addgene no.37825) i.p. using LNPs or LPs at a dose of 10,000 ng of DNA or AAV8 at a dose of 2 x 109 genome copies per mouse (equating to approximately 200 ng of DNA) and serum was collected 3 days later to evaluate ALT levels as described above. Seven days after transfection, mice were euthanized by CO2 inhalation and perfused with 10 mL of Ca2+ /Mg2+ -free HBSS followed by 10 mL of HBSS containing 1 mg/mL type IV collagenase (MP Biomedicals) via the hepatic portal vein.
  • Livers were excised, diced with a scalpel, and incubated for 30 minutes at 37 °C in RPMI containing collagenase at 1 mg/mL and 50 ⁇ g/mL DNAse (MP Biomedicals). Digested liver fragments were gently pressed through a 0.22 ⁇ m mesh filter and the cells were collected, centrifuged at 50 x g for 3 minutes with the supernatant discarded, and then washed twice more with phosphate buffered saline. The remaining cell suspension (50 ⁇ L) from each liver was then moved to polycarbonate tubes and diluted 1:10 in FACS buffer containing anti- mouse CD16/32.
  • Anti-hAAT IgG subclass ratios were assessed as described above, using a single 1:100 sample dilution and the following detection antibodies: goat anti-mouse IgG1-HRP (Abcam ab98693) at 1:10,000, IgG2a-HRP (Abcam ab98698) at 1:5000, IgG2b-HRP (Abcam ab98703) at 1:10,000 and IgG3 (Jackson 115-035-209) at 1:5000. Subclass ratios were calculated by dividing the absorbance of each subclass by that of IgG1 for each individual mouse. [00205] Data Analysis and Statistics [00206] Data were organized and analysed using Graph Pad Prism v.9 for Windows.
  • ALT alanine aminotransferase
  • IL-6 interleukin 6
  • SCr creatinine
  • mice treated with 10 mM TZ9 showed statistically significant increases in ALT and IL-6, with one mouse dying in this treatment group (FIG.55).
  • Visual examination of internal organs at 72 hours revealed significant inflammation and swelling throughout the intestines and abdominal cavity of mice treated with TZ9 at 10 and 20 mM.
  • the toxicity of TZ9 in vivo was unexpected, as in vitro studies indicated TZ9 to be less toxic than TZ3. 39 The discordant results between in vitro and in vivo studies suggest that the cause of toxicity is more complex than simple cytotoxicity, but the exact physiologic mechanism of toxicity is unclear.
  • Lipid based nanoparticles are generally prepared with various lipids to afford a nanoparticle with specific properties, based on the desired application.
  • LPs lipoplexes
  • LNPs lipid nanoparticles
  • TZ3 while successful at encapsulating DNA, trended toward lower encapsulation efficiencies as compared to DOTAP, generally encapsulating 60-70% of DNA vs. DOTAP’s 70-80% encapsulation. While the attributes of the nanoparticles can likely be improved by further altering multiple parameters such as cholesterol content, no additional alterations were made and further evaluation of TZ3 was pursued using PEG550 and DOPE. 79,80 [00213] After optimization in vitro, the PEG550 and DOPE formulation was evaluated in vivo using the same GFP plasmid.
  • TZ3 LNPs 10 ⁇ g of plasmid DNA was transfected into mice and compared with the same dose of DNA delivered via LPs made from a 1:1 ratio of TZ3 and DOPE, previously used for in vitro transfection. 39 Since the resulting nanoparticles were over 200 nm in diameter, they were delivered intraperitoneal (i.p.) based on the concern that intravenous (i.v.) administration could harm the animals. Additionally, previous studies have shown this route to result in similar transfection efficacy as i.v. administration.
  • transfection with LNPs was less efficient than that achieved using an AAV8-GFP vector, carrying the same plasmid, at a dose of 2 x 109 GC per mouse ( ⁇ 200 ng of DNA).
  • transfection with LPs was heterogeneous, mean hepatocyte GFP positivity trended upward over untreated mice.
  • GFP MFI in hepatocytes was significantly increased over untreated mice, while LNP treatment resulted in no increase over baseline (FIG.6D).
  • hAAT transfection was reevaluated, using TZ3 and DOTAP LNPs or LPs.
  • the lipid formulations were made as above and the mice received 10,000 ng of hAAT plasmid DNA.
  • Control mice were given hAAT protein at 25 ⁇ g of protein, calculated on average observed amount of protein reported by Crepso, et al. with liposomal delivery of hAAT plasmid.
  • mice were administered the protein in saline or with 1 mM TZ3, DOTAP, or DMPC.
  • the optimized LP formulation led to detectable hAAT levels in serum in some of the mice (FIG.7), although these were well below the values reported previously for cationic lipid delivery.
  • LP administration led to higher transfection efficiency with average hAAT levels of 9.5 ng/mL for TZ3 and 3 ng/mL for DOTAP LPs, which were closer to those observed in previous work by Crepso, et al. and Ali ⁇ o, et al.
  • CONCLUSION [00222] The present disclosure highlights the utility of cationic triazine lipids as a tool for in vivo research. Evaluation of in vivo toxicity of the compounds showed, surprisingly, that TZ9 confers significant toxicity and mortality via a yet unknown mechanism, which differed from the in vitro toxicity observed during transfection in Example 1. 39 This toxicity not only led to elevations in liver, kidney and inflammatory markers, but also to the death of several animals. However, TZ3 showed comparable toxicity to DOTAP. [00223] The toxicity experiments were followed by evaluation of transfection with TZ3, which demonstrated increased transfection efficacy compared with DOTAP, both in vivo and in vitro.
  • TZ3 serves as a leading candidate for in vivo transfection.
  • LP transfections achieved hAAT levels similar to those reported in previous lipid literature, 57 lipid-based plasmid delivery systems were not able to achieve the levels observed with viral delivery systems. 43, 99 The hAAT plasmid used in these studies is based on a lentiviral system reported by Wilson, et al.
  • Plasmids offer certain advantages over other forms of nucleic acids, including longer stability and lower immunogenicity toward transgenes, 42,44 theoretically making them better suited for long term expression of therapeutic transgenes.
  • formulations containing DOPE and PEG550, rather than DSPC and PEG2000, can enhance the efficacy of plasmid delivery both in cells and in mice.
  • LNPs which contained PEG, reduced titers against the transgene compared with LPs without PEG. While the antibody response to hAAT is relatively low, these data suggest a need for further interrogation of the role of PEG in cationic lipid vaccines.
  • PEG can reduce nanoparticle uptake and transfection in antigen presenting cells (APCs)
  • APCs antigen presenting cells
  • B cells in vivo 104 which could help increase uptake and expression of antigens in B cells that recognize the polymer as an epitope and counter the reduced uptake by phagocytes.
  • Another confounding factor for our evaluation of these findings is that, as reported by Hassett, et al., differences in nanoparticle size can affect titers generated by mRNA vaccines.
  • TZ3 In addition to the modest increase in immunogenicity toward the transgene when delivered as a lipoplex, TZ3 also resulted in robust antibody induction (RET >10 ⁇ 5 (or >100,000)) when used to deliver the hAAT protein. Cationic lipids are known to possess immunomodulatory properties 85,106 and serve as adjuvants, 100 but the significant induction with TZ3 was an unexpected finding. This is particularly notable given that TZ3 induced an antibody response two orders of magnitude greater than DOTAP.
  • Example 3 Triazine-Based Lipids as Vaccine Carriers, Adjuvants, Th1/Th2 Balancers, and NFKB Modulators
  • Standard approaches to generate lipid-based carriers for vaccine design focus on inducing robust immune responses in laboratory animals as measured by increased expression of pro-inflammatory cytokines and antigen-specific antibody responses without a clear understanding of mechanism of action.
  • 108 Efforts to elucidate the mechanism of action of cationic lipids suggest that 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) enhances dendritic cell activation as determined by CD80 expression, with reduced TNF ⁇ expression when stimulated with LPS.
  • DOTAP 1,2-dioleoyl-3-trimethylammonium propane
  • non-canonical NF K B pathway is known to regulate IFN-dependent antiviral immunity, but this pathway is not considered in studies with DOTAP.
  • DOTAP cationic lipids, specifically DOTAP, modulate NF K B through an unknown mechanism which is complicated by the potential for TLR signaling alone or in combination with TLR agonists commonly used in vaccine development.
  • Traditional approaches to vaccine development have focused on activation of the canonical NF K B pathway, which is responsible for broad pro-inflammatory responses leading to variability among patients in both immunity and reactogenicity.
  • cationic substrates are known to modulate the cellular Inhibitor of Apoptosis-1 and 2 (cIAP1 and cIAP2), which are ubiquitin ligases that activate the canonical and inhibits the non-canonical NF K B pathways under basal conditions.
  • cIAP1/2 Upon depletion or inhibition of cIAP1/2 from the cytosol, the canonical pathway is halted and the non-canonical NFKB pathway proceeds resulting in increased antigen presentation, costimulatory molecule expression and reduced reactogenic cytokines (e.g. TNF ⁇ ). Therefore, selective inhibition of the cIAP axis will activate the non- canonical NFKB pathway and enhance vaccine induced immunity without inducing unwanted reactogenicity.
  • cytokines e.g. TNF ⁇
  • the NFKB pathway is composed of a family of transcription factors including p50, p52, p65 (RelA), RelB, and c-Rel that mediate immune activation in the cell.
  • NF K B signaling is also controlled by a series of kinases, ubiquitinases, and inhibitory molecules that regulate nuclear translocation and transcription of NFKB associated genes.
  • 113 NFKB signaling occurs through two pathways, the canonical pathway composed of p50, RelA, and c-Rel, and the tightly regulated and inducible non-canonical pathway composed of p52 and RelB (FIG.59).
  • 116 Canonical stimulation through pattern recognition receptors result in rapid activation and transcription of pro-inflammatory gene signatures (e.g. IL- 6, TNF ⁇ ).
  • pro-inflammatory gene signatures e.g. IL- 6, TNF ⁇
  • the non-canonical pathway is tightly regulated and activated through the TNF receptor superfamily members (e.g. CD40) resulting in germinal center formation, B/T cell interactions, and antibody affinity.
  • TNF receptor superfamily members e.g. CD40
  • Stimulation of the non-canonical pathway is critical for protective vaccine-induced immunity.
  • 112, 142 Targeting NFKB using small molecule modulators has recently been investigated as a method of enhancing antigen specific immune responses while reducing pyrogenic cytokine production.
  • the lynchpin that regulates the canonical and non-canonical pathways are the ubiquitin ligases, cIAP1/2, which activate the canonical pathway and inhibit the non-canonical pathway under basal conditions.
  • cIAP1/2 the ubiquitin ligases
  • vaccine platforms that inhibit cIAP activity will inhibit the canonical NF ⁇ B pathway and promote activation of the non-canonical pathway to enhance the quality of vaccine-induced immunity.
  • Cationic lipids have been shown to limit pyrogenic cytokine expression when stimulated with LPS or TNF ⁇ , while enhancing in vivo immune responses toward antigens.
  • TZ3 demonstrates similar in vivo activity in the absence of additional adjuvants (FIG. 60).
  • DOTAP cationic lipids inhibit the canonical NFKB pathway, while stimulating the non-canonical NFKB pathway.
  • 109,110 The chemistry used to synthesize triazine-based lipids provides an efficient synthetic strategy to achieve compositional diversity to generate structure activity relationships and elucidate its mechanism of action.
  • triazine-based lipids inhibit the canonical NF K B pathway associated with the pro-inflammatory reactogenic response, while stimulating the non-canonical NFKB pathway to improve vaccine induced immunity.
  • Solid-Phase Synthesis of Triazine Lipids Efficient solution based synthetic strategies are based on the thermally controlled chemoselective reactivity of cyanuric chloride. 145, 147, 148 Solution phase reactions rely on protecting groups to prevent side reactions and cross-linking of reactive moieties, which increases the time, materials, and waste generated to prepare the molecules.
  • Cyanuric chloride (trichlorotriazine) is a D3h-symmetric molecule with seemingly equivalent reactivity at each electrophilic carbon.
  • nucleophilic aromatic substitution (SNAr) at any of the carbon centers causes electron density redistribution toward the other two carbons, thereby reducing the reactivity of subsequent reactive sites.
  • SNAr nucleophilic aromatic substitution
  • Increasing the temperature overcomes the reduced reactivity of each carbon moiety to achieve compositional diversity around cyanuric chloride as a linker (FIG.61).
  • the use of commercially available headgroups and tails offers an efficient and inexpensive strategy to generate lipids of diverse composition for continued investigation.
  • lipids Ten of the 12 lipids (TZ1-TZ12) previously synthesized are able to form stable liposomes with a range of transition temperatures (25-65 °C) for lipids containing C18 tails.
  • 145 TZ3 was, in particular, identified to exhibit robust vaccine induced antibody responses and inhibit canonical NF K B signaling, and, as such, served as ideal lipid to synthesize via solid phase synthesis to assess the extent to which a combinatorial lipid library can be readily created via solid phase synthesis using commercially available headgroups and tails.
  • Method of Synthesis Solid-phase synthesis of TZ3 was carried out using the strategy shown in FIG.64.
  • a resin which, in this case, was a 2-chlorotrtityl chloride resin
  • a diamine headgroup or first amine headgroup selected from a library in excess (in this case, for 1h at 0°C) to generate an amine terminated resin for nucleophilic aromatic substitution with cyanuric chloride (in this case, by reacting for 6h at 25°C) to yield a corresponding dichlorotriazine.
  • the resin is thoroughly washed to remove unreacted reagents.
  • a lipid tail selected from a lipid tail library is then added by reacting the dichlorotriazine with the selected lipid tail (in this case, for 6h at 25°C), thus forming a monochlorotriazine with a lipid tail.
  • An additional amine headgroup (or second amine headgroup) is then added to the product yielded from such reaction (i.e., the monochlorotriazine) by reacting the yielded product with another amine selected from a library (in this case, for 12h at 25-80°C).
  • the lipid is cleaved from the resin using mild acid conditions and evaporating the solvent (in this case, 1-5% trifluoroacetic acid in dichloromethane for 3h at 25°C) to produce the final lipid product.
  • the solvent in this case, 1-5% trifluoroacetic acid in dichloromethane for 3h at 25°C
  • the first (diamine) headgroup selected to facilitate the generation of an amine terminated resin was ;
  • the lipid tail selected was ;
  • the second amine headgroup selected following lipid tail addition was .
  • a high yield of TZ3 was synthesized without the need for column chromatography or protecting groups.
  • a library including a variety of additional triazine-based lipids can be created by using alternative first amine headgroups, lipid tails, and/or second amine headgroups from that specified above regarding the synthesis of TZ3.
  • the first (diamine) headgroup may be selected from an amine headgroup library including the lipid tail group may be selected from a tail group library including ;
  • the second amine headgroup may be selected from an amine headgroup library including Using the foregoing libraries of headgroups and lipid tails, a total of 140 different lipids can be synthesized.
  • Triazine-Based Lipids Exhibit Dose Dependent In Vitro Toxicity
  • the toxicity of selected triazine lipids were assessed by lactate dehydrogenase activity as a marker of early apoptosis (FIG.62).
  • Murine bone marrow derived macrophages (BMDMs) were incubated with triazine lipids for 24 hours and exhibit comparable toxicity to commercially available lipids.
  • TZ4, LD50 180 ⁇ M
  • DOTMA Cationic Triazine Lipids Inhibit Canonical NFKB Signaling in Human THP-1 Monocytes
  • THP-1-Blue NFKB monocytes are an ideal tool to investigate the NFKB immunomodulatory potential of compounds.
  • Neutral (DOPE) and anionic lipid TZ2 exhibited no activity.
  • Cationic lipids exhibit immunomodulatory activity in vitro and in vivo. 161, 162 Although the non-canonical pathway and cIAP have not been implicated in the modulation of cationic-lipid vaccine induced immunity, the prior data with DOTAP support the activation of the non-canonical pathway. 109 The signaling cascade that regulates both the canonical and non- canonical NFKB pathways involves cIAP, a ubiquitin ligase, as the major regulator of each cascade.
  • cIAP1 and cIAP2 ubiquitinate scaffolding proteins in the canonical pathway resulting in their degradation, which facilitates IKK ⁇ and IKB ⁇ phosphorylation and subsequent nuclear translocation and of the p65/p50 heterodimer for pro-inflammatory gene transcription.
  • cIAP ubiquitinates the NFKB-Inducing Kinase (NIK), which inhibits the non-canonical NFKB pathway.
  • NIK NFKB-Inducing Kinase
  • TRAF2/3 Upon stimulation through TWEAK/Fn14 signaling, the cIAP binding partners, TRAF2/3, are sequestered at the cell membrane and degraded, which inhibits cIAP activity and prevents ubiqutination of both NFKB pathways resulting in activation of the non-canonical pathway and inhibition of the canonical pathway. 15, 56 Activation of the non-canonical pathway results in dendritic cell maturation, germinal center formation, B/T cell interactions, and protective immunity; however, inclusion of adjuvants that also stimulate the canonical pathway result in unwanted reactogenicity and variable protective immunity. 113 Our current data suggest that TZ3 inhibits the canonical pathway, while stimulating the non-canonical pathway leading to robust antibody responses in vivo.
  • TZ3 provides robust in vivo antibody induction with a model antigen (FIG. 60), 144 that inhibits the canonical NF K B pathway (FIG.63), and increases expression of CD80 (FIG.65) in TZ3 treated macrophages, which is evidence of non-canonical NFKB stimulation.
  • Cationic Triazine Lipids Promote CD80 Expression on Murine Macrophages In Vitro.
  • J774 murine macrophages incubated with TZ3 alone (FIG.65) or co-treatment with TZ3 and LPS (data not shown) for 16 hours result in a dose-dependent increase in expression of the co-stimulatory molecule, CD80.
  • cells are viable across lipid concentrations as determined with ZombieDye live/dead stain.
  • Increased expression of CD80 after TZ3 treatment aligns with data observed for DOTAP, 109 and supports the hypothesis that cationic lipids activate the non-canonical NFKB pathway leading to enhanced antigen presentation and co-stimulatory molecule expression.
  • THP-1-NF K B-Blue cells treated with TZ3 result in a dose dependent inhibition of the canonical NFKB pathway when treated for 16 hours (FIG.63).
  • the duration of treatment in cells prior to stimulation with TNF ⁇ was examined from 1 to 9 hours to determine the temporal effects of cationic lipid treatment.
  • TZ3 was compared to other cationic triazine lipids and DOTAP resulting in similar activity across all timepoints indicating a rapid onset of canonical NFKB inhibition, which indicates TZ3 is most likely modulating NFKB activity through inhibition of a protein target (FIG.66).
  • Modulation of NFKB Signaling Pathways are Monitored in Macrophages Treated with Inhibitors.
  • IKK ⁇ I K ⁇ kinase
  • NF K B signaling can be regulated by a cationic triazine lipid, such as TZ3, through cIAP.
  • BMDC bone marrow derived dendritic cells
  • a cationic triazine lipid such as TZ3
  • cIAP1 and cIAP2 ubiquitin ligases
  • NIK accumulation in treated cells will increase leading to amplified phosphorylation of non-canonical pathway proteins (i.e., IKK ⁇ , p100), while phosphorylation of canonical NFKB proteins (i.e., IKK ⁇ , I K B ⁇ ) will decrease.
  • BMDCs will be treated with TZ3 at the IC80 (10 ⁇ M) introduced to cells as a liposome for different timepoints (5, 15, 30, 45, 60 min) with or without subsequent stimulation with TNF ⁇ .
  • Western blot of protein will be performed using the JESS ProteinSimple Automated Western Blot Analysis System (Bio-Techne) for multiplex fluorescent detection of proteins and phosphoproteins from cell lysates.
  • Cells will be evaluated for cIAP1 and cIAP2 along with canonical NF K B proteins (IKK ⁇ /pIKK ⁇ , I K B ⁇ /pI K B ⁇ , p65, p50) and non-canonical NF K B proteins (NIK, TRAF2/3, IKK ⁇ , RelB, p100, p52) using antibodies specific for each (Abcam, Cambridge, UK).
  • TLR, NOD TLR, NOD
  • Vaccines (Basel) 2021, 9 (3). 138. Vasou, A.; Sultanoglu, N.; Goodbourn, S.; Randall, R. E.; Kostrikis, L. G., Targeting pattern recognition receptors (PRR) for vaccine adjuvantation: From synthetic PRR agonists to the potential of defective interfering particles of viruses. Viruses 2017, 9 (7). 139. Pulendran, B.; P, S. A.; O'Hagan, D. T., Emerging concepts in the science of vaccine adjuvants. Nat Rev Drug Discov 2021, 20 (6), 454-475. 140. Herve, C.; Laupeze, B.; Del Giudice, G.; Didierlaurent, A.

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Abstract

L'invention concerne des lipides triazines et des procédés de synthèse en phase solide pour synthétiser des lipides triazines. Certains lipides triazines décrits peuvent inhiber l'activité transcriptionnelle de NF KB canonique et peuvent ainsi être utilisés dans des vecteurs de transfection non viraux pour une administration avec un antigène immunostimulateur pour réduire la réponse reactogène invoquée par l'antigène immunostimulateur. L'invention concerne également des procédés d'inhibition de l'activité transcriptionnelle de NF KB canonique pendant une réponse immunitaire.
PCT/US2023/027667 2022-07-13 2023-07-13 Lipides triazines, synthèse de lipides et procédés d'inhibition de l'activité transcriptionnelle de nf kb canonique WO2024015531A2 (fr)

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